Identification of in vitro phosphorylation sites in the growth cone protein SCG10. Effect Of phosphorylation site mutants on microtubule-destabilizing activity

SCG10 is a neuron-specific, membrane-associated protein that is highly concentrated in growth cones of developing neurons. Previous studies have suggested that it is a regulator of microtubule dynamics and that it may influence microtubule polymerization in growth cones. Here, we demonstrate that in vivo, SCG10 exists in both phosphorylated and unphosphorylated forms. By two-dimensional gel electrophoresis, two phosphoisoforms were detected in neonatal rat brain. Using in vitro phosphorylated recombinant protein, four phosphorylation sites were identified in the SCG10 sequence. Ser-50 and Ser-97 were the target sites for protein kinase A, Ser-62 and Ser-73 for mitogen-activated protein kinase and Ser-73 for cyclin-dependent kinase. We also show that overexpression of SCG10 induces a disruption of the microtubule network in COS-7 cells. By expressing different phosphorylation site mutants, we have dissected the roles of the individual phosphorylation sites in regulating its microtubule-destabilizing activity. We show that nonphosphorylatable mutants have increased activity, whereas mutants in which phosphorylation is mimicked by serine-to-aspartate substitutions have decreased activity. These data suggest that the microtubule-destabilizing activity of SCG10 is regulated by phosphorylation, and that SCG10 may link signal transduction of growth or guidance cues involving serine/threonine protein kinases to alterations of microtubule dynamics in the growth cone.     

A noncompetitive peptide inhibitor of the nicotinic acetylcholine receptor from Conus purpurascens venom

A paralytic peptide, psi-conotoxin Piiie has been purified and characterized from Conus purpurascens venom. Electrophysiological studies indicate that the peptide inhibits the nicotinic acetylcholine receptor (nAChR). However, the peptide does not block the binding of alpha-bungarotoxin, a competitive nAChR antagonist. Thus, psi-conotoxin Piiie appears to inhibit the receptor at a site other than the acetylcholine-binding site. As ascertained by sequence analysis, mass spectrometry, and chemical synthesis, the peptide has the following covalent structure: HOOCCLYGKCRRYOGCSSASCCQR* (O = 4-trans hydroxyproline; * indicates an amidated C-terminus). The disulfide connectivity of the toxin is unrelated to the alpha- or the alphaA-conotoxins, the Conus peptide families that are competitive inhibitors of the nAChR, but shows homology to the mu-conotoxins (which are Na+ channel blockers).     

Cathepsin E expression by normal and premalignant cervical epithelium

We have investigated the expression of the aspartic proteinase cathepsin E and HLA-DR and the presence of HPV16 in normal squamous epithelium (n = 8) and low-grade (n = 21) and high-grade (n = 14) intraepithelial squamous lesions of the uterine cervix. Immunohistochemistry of cervical biopsies revealed that up-regulation of cathepsin E expression was related to increasing severity of the cervical intraepithelial neoplasia (CIN). Up-regulation of protein was associated with increased message as assessed by in situ hybridization. Langerhans cells and the majority of koilocytes did not express detectable cathepsin E levels. Although there was also an up-regulation of HLA-DR expression by cervical keratinocytes in cervical intraepithelial neoplasia lesions, as determined by immunohistochemistry, no significant correlation was found between HLA-DR and cathepsin E expression in these lesions; neither was expression of cathepsin E correlated to the presence of HPV16, detected by polymerase chain reaction. The expression of cathepsin E, an aspartic proteinase that is reported to play a role in antigen processing for presentation by class II major histocompatibility complex molecules, is associated with cellular dedifferentiation in cervical intraepithelial neoplasia.     

Cure of malignant ascites and generation of protective immunity by monoclonal antibody-targeted activation of a glucuronide prodrug in rats

We examined the in vivo efficacy of targeting beta-glucuronidase (betaG) to activate a glucuronide prodrug (BHAMG) of p-hydroxyaniline mustard (pHAM) at hepatoma ascites in Sprague-Dawley rats. Injection i.p. of 500 microg RH1-betaG, a conjugate formed between recombinant betaG and monoclonal antibody RH1 with specificity for an antigen expressed on AS-30D rat hepatoma cells, into rats bearing AS-30D ascites resulted in the accumulation of 54 microg conjugate per 10(9) tumor cells after 2 hr. Ascites fluid and serum contained 0.53 and 0 microg/ml, respectively, RH1-betaG 2 hr after injection of the conjugate. Conjugate binding to AS-30D cells was heterogeneous and non-saturated, as determined by flow cytometry. BHAMG was less toxic than pHAM to SD rats based on measures of animal mortality, weight loss and hematological toxicity. Treatment of rats bearing established hepatoma ascites with 500 microg RH1-betaG followed 2 hr later with a single i.p. injection of 30 mg/kg BHAMG or 3 i.p. injections of 10 mg/kg BHAMG 2, 3 and 4 hr later resulted in the cure of 6/8 and 8/8 animals, respectively. Treatment with BHAMG or pHAM alone did not produce cures, whereas treatment with a control antibody-betaG conjugate and BHAMG produced significantly greater hematological toxicity compared to treatment with RH1-betaG and BHAMG. All cured rats were completely protected from rechallenge with 2 x 10(7) AS-30D cells, indicating that successful treatment of animals induced protective immunity.     

A linkage map of human chromosome 21:43 PCR markers at average intervals of 2.5 cM

A genetic linkage map of human chromosome 21q (HC21q) containing 43 markers genotyped by the polymerase chain reaction in the CEPH pedigrees is presented. The markers placed on this map are highly polymorphic with an average heterozygosity of 61%. The average interval size of the markers localized at 1000:1 odds is 2.5 cM. The map has a total length of 65.5 cM, with male and female lengths of 47.7 and 83.3 cM, respectively. The genotypes used in the construction of this map were subjected to rigorous error checking, which is reflected in the shorter map length compared to previous maps; the estimated error rate in genotyping is less than 0.04%. As noted in previous linkage maps there is increased recombination in females on proximal HC 21q and in the male in a region near the telomere. This map of HC 21 represents a highly informative and dense meiotic linkage map and will be useful in linking disease phenotypes to loci on this chromosome.     

Stochastic model for a wavelike exothermal reaction in condensed heterogeneous systems

The labile protons of two 32-base-pair, four-arm models of immobile Holliday junctions have been studied by two-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy. Overlap of resonances in the imino-imino region of two-dimensional nuclear Overhauser enhancement (NOE) spectra necessitates the use of a multi-pathway approach for obtaining sequence-specific assignments wherein all possible NOE connectivities to the labile protons are utilized, including those from the 2H of adenine, 5CH3 of thymine, and 5H of cytosine. Resonance assignments are obtained for all slowly exchanging imino and cytosine amino protons. Base-pairing up to and including the junction point is found in all four arms of both Holliday junctions. Several cross-arm NOE connectivities are identified and can be used to infer the geometry of the helical stacking domains. The two Holliday junctions studied, which differ only by the exchange of two base pairs at the branch point, appear to have opposite arm stacking geometries. These assignments form an important part of the critical background for detailed NMR analysis of Holliday junction structure and dynamics.     

Patient preference for self-collected cultures for group B streptococcus in pregnancy

The purpose of this study was to examine the factors which affect the level of fatigue among patients participating in clinical trials in which this symptom had been assessed with the EORTC QLQ-C30. Data were assembled from 2390 patients in ten clinical trials in which the QLQ-C30 had been used to assess baseline and on-study quality of life. The relationship between the level of fatigue reported by the patients on the fatigue scale of this questionnaire and patient and disease characteristics was assessed in univariate and multivariate cross-sectional analyses. In addition, changes in fatigue scores were compared in a longitudinal analysis among patients on two arms of an anti-emetic trial whose emesis control was markedly different. Baseline fatigue levels differed substantially among patients taking part in the different trials. Factors associated with greater fatigue severity on univariate analysis included: female gender, presence of metastatic disease, and poorer performance status. In addition, on multivariate analyses the oldest patients were found to have less fatigue, as were patients with breast cancer, while patients with ovarian and lung cancer experienced greater fatigue. Patients on the arm of the anti-emetic trial in which emesis was better controlled showed significantly less increase in fatigue after receiving chemotherapy. The fatigue scale of the QLQ-C30 appears to provide a useful approach to assessing this important symptom. The relationships found between fatigue and patient and disease characteristics need further exploration as does the degree to which the QLQ-C30 fully captures this dimension of quality of life.